Diagnostic assays using antibodies and those using nucleic acids have been found to be useful for detection or determination of very low concentrations of various medically, environmentally or industrially important organic substances (e.g., antigens, antibodies, DNA, RNA, etc.) in liquid media. Nowadays, clinical tests or diagnostic test kits which have resulted from the embodiment of these methods are practically used because the tests can be rapidly and readily conducted by using them.
When using such a kit, usually, a body fluid containing an unknown amount of an organic substance to be tested is bound to a solid phase or other chemical substance (e.g., an enzyme-labeled antibody, etc.). In many cases, the solid phase used for the kit has an immobilized antigen (hapten), antibody or nucleic acid on its surface, and such a substance takes part in a competitive binding reaction or binding pair formation reaction with the above organic substance or other chemical substance. Usually, this binding reaction is conducted prior to a reaction for detection or determination, i.e., a reaction for generating a signal such as light, electric current or the like in a measurable amount which varies in proportion to the concentration of a substance to be tested (usually, mediated by an enzyme which correlates to concentration of a substance to be tested as described hereinafter).
In many cases, an enzymatic reaction is employed to generate a signal because amplification can thus be utilized. Usually, determination of the amount of an enzyme is conducted by maintaining the enzyme under specific incubation conditions (including pH, salt, temperature, substrate, composition of buffer solution, etc.) for a certain period of time, and then measuring the product of the enzymatic reaction.
In a most sensitive method, after separation or washing of an enzyme bound to a solid phase carrier, the enzyme is incubated in a specific medium to cause an enzymatic reaction to generate light as a signal (photosignal). Under these conditions, sensitivities are often limited due to limitation of the catalytic reaction rate of the enzyme or the apparatus used to measure the enzymatic activity. The sensitivity of enzyme detection is of importance because it defines the sensitivity of detection of a substance to be tested [see Ekins et al., J. Biolum. Chemilum., 4, 59-78 (1989)]. Therefore, improvement in the sensitivity of detection of an enzyme label has been desired and various studies are directed to this topic.
Further, there are problems and limitations peculiar to respective types of the conventional measurements of enzymes. For example, an enzyme which catalyzes a redox reaction is sensitive to substances which chemically interfere with the reaction such as ascorbate, sulfhydryl compounds, iron and the like. In an enzymatic reaction, it is necessary to maintain the concentration of such interfering substances as low as possible, and various attempts to remove the interfering substances or to minimize their influence thereof have been made to improve this kind of technique. For example, Scott and Kricka [Bioluminescence and Chemiluminescence, 237-240 (1987)] disclose an improved method for purifying the substrate luminol which is subjected to an enzymatic reaction with horseradish peroxidase (hereinafter sometimes abbreviated as HRP) to generate a photo-signal. However, even in the case of recrystallized luminol, a trace amount of impurities remain. In addition, impurities may be formed during storage of an aqueous solution of luminol. These impurities lower the sensitivity of enzyme detection. Furthermore, there is a problem that interfering substances are still present in impurities from other reaction components such as an enhancer used for enhancing the reaction of an enzyme with luminol, an organic solvent used for dissolving an enhancer, a buffer salt, substances absorbed on the surface of a reaction vessel and the like. Particularly, an enzyme which releases free radicals by reaction with an enhancer or the like is liable to be influenced by the above interfering substances. Namely, substances which quench free radicals in a solution to be subjected to readout, for example, impurities such as protein, other sulfhydryl compounds, various amino acids, phenols and quinones, readily react with free radicals to interfere with the measurement. Therefore, such an enzyme is very likely to undergo interference from the above substances. Another disadvantageous result caused by interference from the above substances is time lag. This time lag corresponds to a period of time required for consuming free radicals produced by reaction of an enzyme with interfering substances and, a chain reaction may be firstly initiated by free radicals produced after all the interfering substances have been consumed by reaction with free radicals [Morrison and Boyd, "Organic Chemistry", page 50 (1973)].